A new technology platform for studying protein function

研究蛋白质功能的新技术平台

• 批准号: 7387091
• 负责人: MATTHEW P DELISA
• 金额: $ 19.13万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-09 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7387091
• 关键词: Acquired Immunodeficiency Syndrome; Affinity; Antibodies; Antigens; Antisense RNA; Arginine; Bacteria; Binding; Biological Assay; Biological Process; Categories; Cell Nucleus; Cells; Class; Cytoplasm; Degenerative Disorder; Development; Disease; Dreams; Drug Delivery Systems; Engineering; Environment; Exhibits; Gene Deletion; Gene Expression; Generations; Genes; Goals; Half-Life; Human; Immunoglobulins; In Vitro; Knock-out; Knowledge; Libraries; Life; Malignant Neoplasms; Mammalian Cell; Mediating; Methods; Molecular; Monoclonal Antibodies; One-Step dentin bonding system; Outcome; Pathway interactions; Pharmaceutical Preparations; Phenotype; Play; Process; Property; Proteins; Proteome; Proteomics; Quality Control; RNA; RNA Interference; Range; Reagent; Research; Role; Scheme; Specificity; Structure; System; Technology; Testing; Time; Twin Multiple Birth; Validation; Work; bacterial genetics; base; biological research; cellular targeting; combinatorial; design; disulfide bond; extracellular; functional genomics; genetic selection; in vivo; innovation; interest; new technology; novel; protein function; research study; tool

DESCRIPTION (provided by applicant): Ever since the invention of monoclonal antibodies in 1975 and, more recently, the development of various in vitro antibody display technologies, antibodies have become one of the most powerful tools in biological research and are presently the fastest growing category of new drug entities. One molecular format that shows great promise is the intracellular antibody or intrabody that exploits the specificity and diversity of immunoglobulins to target a wide range of intracellular proteins by expressing the antibody in vivo. In principle, whatever can be achieved by a monoclonal antibody in the extracellular environment can be similarly achieved inside of a cell using an intrabody. Since intrabody synthesis can be constitutive or inducible, the level of inactivation can be toggled which might allow for a wider range of phenotypes than can be observed with gene deletion, antisense or RNAi-based knockdown strategies. Further, since intrabodies are proteins, they possess a much longer half-life compared to RNA and are also more specific to their target molecules. Also, it is possible to design or engineer intrabodies to block certain domains of a particular target protein, thus allowing for the decoupling of multiple protein activities of a single target. This might prove particularly useful for essential targets that have more than one cellular activity. Finally, since intrabodies can be multivalent, simultaneous functional knockout of two or more cellular targets is possible. Based on the above features, intrabodies are expected to play an important and immediate role for target identification and validation in functional genomics and/or proteomics. The long-term objective of this research effort is to develop a proteome-wide repertoire of intrabodies for probing and modulating protein activities inside living cells. The objective of this particular application, which is the first step towards our long-term goal, is to create a novel platform technology based on the bacterial twin-arginine translocation (Tat) pathway that enables rapid, one-step genetic selection of single-chain intrabodies against virtually any intracellular target protein. To accomplish the overall objective of this application, the following specific aims are proposed: (1) develop a genetic selection based on unique mechanistic features of the bacterial Tat system for isolating intrabody-antigen pairings; and (2) engineer intrabodies that specifically inhibit biological processes. Intrabodies are an emerging class of antibody molecules that function (e.g., bind their cognate antigen) intracellularly and, owing to their specificity and diversity, have the potential to block, suppress, alter or even enhance a vast array of biological processes. Therefore, the focus of these studies is to develop a technology platform for rapid, large-scale synthesis of intrabodies that could be used as (i) functional genomics reagents that enable characterization of novel gene products and validation of these gene products as potential drug targets and (ii) drug entities that be used in the treatment of human disorders such as cancer, AIDS or neuro-degenerative disorders.

描述（申请人提供）：自1975年单克隆抗体发明以来，以及最近各种体外抗体展示技术的发展，抗体已成为生物研究中最强大的工具之一，也是目前增长最快的类别新药物实体。一种显示出巨大前景的分子形式是细胞内抗体或胞内抗体，它利用免疫球蛋白的特异性和多样性，通过在体内表达抗体来靶向多种细胞内蛋白质。原则上，无论单克隆抗体在细胞外环境中能够实现什么，都可以使用胞内抗体在细胞内部类似地实现。由于体内合成可以是组成型或诱导型，因此可以切换失活水平，这可能允许比通过基因删除、反义或基于 RNAi 的敲低策略观察到的更广泛的表型。此外，由于胞内抗体是蛋白质，因此与 RNA 相比，它们的半衰期要长得多，而且对其靶分子也更具特异性。此外，还可以设计或改造胞内抗体来阻断特定靶蛋白的某些结构域，从而实现单个靶标的多种蛋白活性的解耦。这对于具有多种细胞活性的重要靶标可能特别有用。最后，由于胞内抗体可以是多价的，因此可以同时功能性敲除两个或多个细胞靶标。基于上述特征，胞内抗体有望在功能基因组学和/或蛋白质组学中的靶标识别和验证中发挥重要而直接的作用。 这项研究工作的长期目标是开发蛋白质组范围内的抗体库，用于探测和调节活细胞内的蛋白质活性。这一特定应用的目标是实现我们长期目标的第一步，是创建一种基于细菌双精氨酸易位 (Tat) 途径的新型平台技术，该技术能够快速、一步地遗传选择单链精氨酸。针对几乎任何细胞内靶蛋白的链内抗体。为了实现本申请的总体目标，提出以下具体目标：（1）开发基于细菌Tat系统独特机制特征的遗传选择，用于分离体内抗原配对； (2) 设计专门抑制生物过程的体内抗体。胞内抗体是一类新兴的抗体分子，其在细胞内发挥作用（例如，结合其同源抗原），并且由于其特异性和多样性，有可能阻断、抑制、改变甚至增强大量的生物过程。因此，这些研究的重点是开发一个快速、大规模合成体内抗体的技术平台，该平台可用作（i）功能基因组学试剂，能够表征新的基因产物并验证这些基因产物作为潜在的药物靶点(ii) 用于治疗人类疾病如癌症、艾滋病或神经退行性疾病的药物实体。